The long-term objective of this laboratory is to understand the mechanisms of antiarrhythmic drug modification of the cardiac sodium channel. The specific goals of this proposal continue to pursue those objectives by using an experimental approach that combines site-directed mutagenesis of the human heart Na channel (hH1, Nav 1.5) and the recording of gating currents (electrical signals that result from the movement of the channel's voltage sensors reflecting its molecular conformational states) with targeted modification of Na channels by local anesthetic drugs, peptide toxins, and methanothiosulfonate (MTS) agents. During the current grant period we have applied this approach and have made several very exciting findings that point to an overall understanding of the basis of use-dependent block of Na current by lidocaine and quaternary amines. In this grant application we propose to expand upon our previous work with new studies on cardiac Na channels that will test our hypotheses on use-dependent block by lidocaine and on the interactions of voltage sensors with channel inactivation. The experiments will use both wild type and site-directed mutated cardiac Na channels heterologously expressed in tissue-cultured mammalian cells. 1. Determine if the voltage sensors from domains I & II each contribute about 20% to the maximal gating charge (Qmax) of the cardiac Na channel. 2. Determine if the voltage sensor from domain III has a major role in closed-state inactivation while the voltage sensors in domains I, II & IV have little or no role in closed-state inactivation in wild-type cardiac Na channels. 3. Determine if use-dependent block by lidocaine primarily results from the voltage-dependent conformational position of the voltage sensor in domain III. 4. Determine if the local anesthetic drug binding site located in the pore formed by the S6 segments is allosterically coupled to the stabilization of the voltage sensor in domain III by the S4-S5 linker in domain III. 5. Determine if the effective gating charge contribution to Qmax by the voltage sensor in domain IV is dependent upon the polypeptide backbone length of its S3-S4 linker. The results of these studies should improve our overall understanding of the molecular mechanisms of anti-arrhythmic drugs modification of cardiac Na channels. [unreadable] [unreadable]